Development of a validated UPLC-MS/MS method for quantification of p38 MAPK inhibitor PH-797804: application to a pharmacokinetic study in rat plasma
Jianyuan Qu, Chunling Zhou, Nan Hao, Guangliang Chen, Shuyue Xia, Hongjun Wei, Lina Fang
PII: S1570-0232(19)31355-8
Reference: CHROMB 121877

To appear in: Journal of Chromatography B

Received Date: 9 September 2019
Revised Date: 21 October 2019
Accepted Date: 8 November 2019

Please cite this article as: J. Qu, C. Zhou, N. Hao, G. Chen, S. Xia, H. Wei, L. Fang, Development of a validated UPLC-MS/MS method for quantification of p38 MAPK inhibitor PH-797804: application to a pharmacokinetic study in rat plasma, Journal of Chromatography B (2019), doi:

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© 2019 Published by Elsevier B.V.

Development of a validated UPLC-MS/MS method for quantification of p38 MAPK inhibitor PH-797804: application to a pharmacokinetic study in rat plasma

Jianyuan Qua, Chunling Zhoub, Nan Haoa, Guangliang Chena, Shuyue Xiac,

Hongjun Wei d, Lina Fang de*

aCollege of Basic Medical Science, Shenyang Medical College, Shenyang 110034,


bLiaoning Institute for Drug Control, Liaoning Inspection, Examination & Certification Centre, Shenyang 110035, China
cRespiratory Medicine Department, Central Hospital Affiliated to Shenyang Medical College, Shenyang 110034, China
dTian jin JF-Pharmaland Technology Development Co., Ltd, Tianjin, 300457, China

eCollege of Pharmacy, Shenyang Medical College, Shenyang 110034, China

*Corresponding author at: College of Pharmacy, Shenyang Medical College, Shenyang 110034, China.
E-mail address: [email protected] (L.N. Fang)


PH-797804 is a selective p38 MAPK inhibitor currently evaluated in clinical trials. This study described a validated UPLC-MS/MS combined with one-step protein precipitation extraction method for determination of PH-797804 in rat plasma. After protein precipitation with acetonitrile, the plasma sample was analyzed by a Waters Acquity UPLC BEH C18 column, with acetonitrile/0.1% formic acid (70:30) as the mobile phase. Mass spectrometric detection was conducted with a Waters TQ-S mass spectrometer via electrospray, positive-mode ionization, with target quantitative ion pairs of m/z 476.895→126.860 for PH-797804, and 482.726→269.707 for regorafenib (internal standard). The assay showed a good linearity over the range of 1.0-1600 ng/mL, with acceptable accuracy (RE from -7.8% to 8.5%) and precision (RSD within 8.4%) values. Recovery from plasma was 81.4-90.2% and matrix effect was negligible (93.3-95.4%). The validated method presented a quantification method of PH-797804 in detail for the first time and utilized for a pharmacokinetic study at three dose concentrations after oral administration in Wistar rats. The pharmacokinetic profiles of PH-797804 showed a linear relationship between drug concentration and dose, which provided dosage and safety information on further clinical studies.

Keywords: PH-797804; p38 MAPK inhibitor; UPLC-MS/MS; pharmacokinetic study

⦁ Introduction

p38 kinase is a critical enzyme of the mitogen-activated protein kinase (MAPK) family, which modulates the production and signaling of inflammatory cytokines such as tumor necrosis factor-α, interleukin-1 and interleukin-6 [1, 2]. By inhibition of the expression of inflammatory cytokines and downstream signaling via multiple cytokines, p38 kinase inhibitors have shown significant effects in rheumatoid arthritis, cytokine-mediated disease, and other inflammation related diseases [3-6].
PH-797804 is a selective p38 MAPK inhibitor, which could block the production of inflammatory cytokines and decrease its activity [7-9]. PH-797804 had been widely used in the treatment of various clinical trials of inflammatory diseases, including chronic obstructive pulmonary disease [10], osteoarthritis [11], and right ventricular hypertrophy [12], which all showed good efficacy and safety in the patients. A clinical trial (NCT 00559910) in patients with Chronic obstructive pulmonary disease (COPD) demonstrated PH-797804 improved lung function parameters as well as dyspnea [13]. In addition, PH-797804 also showed efficacy in breast cancer cells [14] and protection from simian immune deficiency (SIV) -mediated immune system deterioration in vivo [15].
It was known that pharmacokinetics can explain and predict a variety of events related to the efficacy of drugs. Although PH-797804 has been tested in clinical trials, there is no analytical method of PH-797804 in detail has been reported. In this study, a rapid and sensitive UPLC-MS/MS combined with one-step protein precipitation extraction method has been established and validated for the determination of PH-797804 in rat plasma. Specificity, linearity, accuracy, precision, extraction

recovery, matrix effect and stability were fully validated which demonstrated the robustness of our method. The pharmacokinetic profiles after oral administration of PH-797804 at three different doses were investigated. The results may be utilized for the application to the clinical study of PH-797804.
⦁ Materials and methods

⦁ Materials and reagents

PH-797804 (purity > 99.1%, Fig. 1A) and regorafenib (BAY73-4506, purity > 98.8%, internal standard, IS, Fig. 1B) were obtained from Shanghai AZBIOCHEM Biotechnology Co., Ltd (Shanghai, China). All of the methanol, acetonitrile and formic acid were of HPLC grade and provided by Fisher Scientific (Fair Lawn, NJ, USA). Distilled water was used in the experiment.
⦁ Instruments and UPLC–MS/MS conditions

Chromatographic separation was conducted on an Acquity UPLC system (Waters, USA), using an Acquity UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm, Waters, USA). The mobile phase included phase A, acetonitrile, and phase B, 0.1% formic acid in water (70:30) at an isocratic flow rate of 0.20 mL/min. The column temperature was set at 35°C.
Samples were analyzed on a Waters TQ-S mass spectrometer (Waters, USA). The detection was performed via electrospray, positive-mode ionization, with target quantitative ion pairs for PH-797804 and the internal standard (IS) were m/z 476.895→126.860, 482.726→269.707, respectively. Other optimal parameters were

as follows: curtain voltage, 3.0 kV; desolvation temperature, 350°C; desolvation gas flow: 800 L/Hr.
⦁ Standard solution and QC samples

Calibration and QC stock solutions of PH-797804 were separately dissolved in methanol at a final concentration of 1.0 mg/mL. The calibration standard solutions for PH-797804 were diluted to obtain a range from 10 to 16000 ng/mL with methanol. Calibration standard solutions of PH-797804 were diluted in rat plasma to obtain calibration standard samples (1.0, 5.0, 25, 100, 400, 800, 1600 ng/mL). QC samples at low, medium, high concentrations (2.5, 100, 1200 ng/mL) were produced independently. The working solution of the IS was prepared in methanol to get a final concentration of 500 ng/mL.
⦁ Sample preparation

A volume of 100 μL of plasma sample was spiked with 50 μL of the IS solution and mixed for 30 s. After adding 400 μL of acetonitrile, samples were mixed for 1 min and then centrifuged for 5 min at 8500 rpm. 2 μL of supernatant was used for analysis after filtering.
⦁ Method validation

⦁ Calibration curve and LLOQ

The linearity of the calibration curve was measured in triplicate to establish the calibration range. The correlation coefficient (r) should be at least 0.99, with the

acceptable accuracy of the LLOQ less than ±20% while the other concentration points less than ±15%.
⦁ Specificity and carry-over

To study whether there were endogenous matrix constituents or potential interferences, six different batches of drug-free plasma and spiked samples at the LLOQ were produced and analyzed. After injection of the ULOQ, a blank sample was injected to investigate the carry-over, where the peak areas generated from blank sample had to be < 20% of the LLOQ of the analyte and < 5% of the IS with six replicates [16].
⦁ Accuracy and precision

Six QC sample replicates at three concentration levels were assessed on three separate occasions to evaluate the accuracy and precision of the method. A mean accuracy (RE) of ±15% was accepted, and the precision (RSD) had to be <15%.
⦁ Recovery and matrix effect

The extraction recovery and matrix effect of PH-797804 were evaluated in samples of six different subjects at three QC levels according to US FDA guidelines and previous studies [17, 18]. The recovery was obtained by comparing peak area ratios of analytes in extracted samples with those in post-extracted spiked samples. The matrix effect was assessed by determining the peak areas of the analytes in post-extracted spiked samples to those acquired from unextracted samples.

⦁ Dilution integrity

Experiment for assessing the dilution integrity of the method was conducted as followed. Six spiked plasma sample with a concentration of 4000 ng/mL (2.5 times of ULOQ) was prepared by adding a standard solution in blank plasma, then diluted 2.5-fold with blank plasma to achieve a concentration within the linear range [19]. To be specific, 60 μL of blank plasma was added into 40 μL of spiked plasma (4000 ng/mL) to generate a diluted sample (1600 ng/mL), and then further processed as described in sample preparation and analyzed. The result was acceptable when the accuracy (RE) and precision (RSD) within ±15%.
⦁ Stability

The stability of PH-797804 was evaluated at three QC levels under four different storage conditions: 30 days at -20°C, after three freeze-thaw cycles, 12 h at room temperature, and samples after preparation at 4°C for 12 h. The acceptable criteria of the stability had to be within ±15%, with precision less than 15%.
⦁ Pharmacokinetic application

Male SPF grade Wistar rats (220-250 g) were provided by Liaoning Changsheng Biotechnology co., Ltd, and fed in an environmentally controlled room. After acclimatized, 18 rats were randomly divided into three groups and received different dosage treatments (PH-797804 was dissolved in DMSO, and suspended with 0.5% CMC-Na to get three concentration levels at 10, 20 and 40 mg/kg, respectively). Blood from the suborbital vein (about 0.4 mL) were collected during the does interval

(0, 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12 and 24 h), and then centrifuged and separated immediately before storage.
The plasma concentrations of PH-797804 at different timepoints were presented as mean ± SD. Pharmacokinetic parameters including Cmax, tmax, t1/2, CL/F, Vz/F, AUC and AUMC were calculated under a noncompartmental model with Phoenix WinNonlin software (version 5.2).
All analyses were performed with SPSS software 19.0. The statistical differences of pharmaceutical parameters were analyzed as follows: tmax and t1/2 were calculated with Kruskal-Wallis test, CL/F and Vz/F were calculated by one-way analysis of variance, when p < 0.05 would be considered as statistically significant.
⦁ Results

3.1 Method development

The chromatographic conditions optimized were the mobile phase composition and injection volume to achieve efficient separation and resolution from endogenous components of plasma and to obtain sharp peak shape as well as adequate peak responses in short run time.
We tested methanol and acetonitrile as the mobile phase for the analytes. Compared with methanol, acetonitrile resulted in better peak shape for PH-797804. After addition of formic acid, we achieved a good peak shape and increase ion intensity of PH-797804 in positive ionization mode. The total retention time can not be further shortened with a larger proportion of the organic phase as the mobile phase, because a strong matrix

effect can be observed at the same time. Finally, a mobile phase condition, composed with acetonitrile and 0.1% formic acid in water (70:30) was adopted and the total run time was 3.0 min. 2 μL of supernatant was used for injection after filtering.
The optimization of mass conditions for PH-797804 and the IS were investigated in both positive and negative modes. To obtain a better mass response, positive ionization mode was selected because both of the analytes are nitrogenous compounds. Parameters including ion transition, cone voltage (40 V for PH-797804, 64 V for the IS) and collision voltage (38 V for PH-797804, 36 V for the IS) were determined with automatic optimization procedure.
⦁ Method validation

⦁ Calibration curve and LLOQ

The calibration curves for the quantification of PH-797804 in rat plasma were linear over the range of 1.0-1600 ng/mL in three consecutive days. The correlation coefficients (r) were not less than 0.9965 with a representative linear regression equation of y = 0.1099 x + 0.00576 for PH-797804. The LLOQ was 1.0 ng/mL with good accuracy and precision (RE: -4.6%, RSD: 6.3%).
⦁ Specificity and carry-over

PH-797804 and the IS were detected at retention times of 1.51 min and 2.06 min, respectively. There was no significant co-elution interference being detected at the same times, which proved a good selectivity. Representative chromatograms of blank

plasma sample, spiked sample with PH-797804 at LLOQ (1.0 ng/mL) and the IS, rat plasma sample 0.75 h after dosing of PH-797804 were presented in Fig. 2.
The carry-over of the ULOQ for the analyte was less than 12.6% of the LLOQ and

< 4.1% for the IS.

Accuracy and precision

Data of accuracy and precision of the method were listed in Table 1. The range of quality control (QC) samples based accuracy was from -7.8% to 8.5% for PH-797804, and the intra- and inter-day precisions for PH-797804 were no more than 8.4%. The results all met the acceptable criterion (RE within ±15%, RSD<15%), which demonstrated the accuracy and reliability of the developed method.
⦁ Recovery and matrix effect

The mean extraction recoveries of PH-797804 were all more than 81.4% at three QC levels, and the recovery of IS was 82.2 ± 5.9%. No significant matrix effects were found in the test samples. In a word, the results of the recovery and matrix effect for PH-797804 and the IS were all within the acceptable criterion (Table 1).
⦁ Dilution integrity

Diluted samples (concentration above the ULOQ) with six replicates were measured after diluting the concentration of 4000 ng/mL to 1600 ng/mL (2.5 fold), and the accuracy was 96.0 ± 6.7%.

⦁ Stability

Assessment of stabilities of the QC samples was conducted at three concentration levels under four different circumstances. The results demonstrated good stabilities at
-20°C for 30 days, after three freeze and thaw cycles, 12 h at room temperature, and at 4°C in autosampler for 12 h after preparation (Table 2).
⦁ Pharmacokinetic application

A pharmacokinetic study of PH-797804 was carried out to prove the applicability of the established UPLC-MS/MS method. A single 10, 20 or 40 mg/kg dosage of PH-797804 was administrated to adult male Wistar rats in the present study. The plasma samples in high dose group showing concentrations over the range of calculation curve were further diluted 2.5-fold with blank plasma to achieve a concentration within the linear range, and then prepared and analyzed in accordance with the method established above. The mean plasma concentration-time curves of PH-797804 were presented in Fig. 3, and the main pharmacokinetic parameters were summarized in Table 3.
As shown in Table 3, tmax were located as 2.50, 4.00 and 3.67 h, and Cmax were detected as 0.65 ± 0.24, 1.24 ± 0.26 and 2.34 ± 0.42 μg/mL at dosage of 10, 20 and 40 mg/kg, respectively. No statistically differences in the main pharmacokinetic parameters (tmax, t1/2, Vz/F and CL/F) of PH-797804 were observed within groups at different doses. In addition, data of Cmax, AUC0→24h, AUC0→∞, AUMC0→24h and AUMC0→∞ were increased with the administration dosage increasing, and regression

analysis results showed that Cmax, AUC0→24h, AUC0→∞, AUMC0→24h and AUMC0→∞ were linear with the dosage with correlation coefficients (r) more than 0.81, respectively.

In the present work, we reported a validated method for the quantitation of PH-797804, a selective p38 MAPK inhibitor in rat plasma. As far as we know, this is the first analytical method reported in detail for determination of PH-797804 in biological specimen. The simple sample preparation approach together with the short analytical time (3.0 min) make the present method excellently suitable for pharmacokinetic study.
Protein precipitation was tested with acetonitrile and methanol as the extraction solvents. The recoveries with both of the solvents were more than 80%. Compared to methanol, acetonitrile achieved a smooth baseline which resulted in a lower S/N of baseline and better sensitivity. This one-step protein precipitation extraction procedure made the sample preparation fast and easy, and suitable for pre-treating a large number of samples.
Regorafenib was employed as the IS because it had a similar molecular mass and chemical structure units with PH-797804. Meanwhile, both of them exhibited similar characteristics in chromatographic behavior, ion signal intensity as well as extraction recovery.
According to the pharmacokinetic results, there was a linear relationship between plasma drug concentration and dosage, indicated the safety of administration within the

dose range (10-40 mg/kg). The pharmacokinetic profiles of PH-797804 could provide dose and safety information on further clinical studies.
⦁ Conclusions

A sensitive and fast UPLC-MS/MS method has been established and utilized for a pharmacokinetic study of PH-797804 after oral administration at three dose levels in rats. The pharmacokinetic profiles of PH-797804 showed a linear relationship between drug concentration and dose. The validated assay and the results may offer useful information on clinical studies of PH-797804, which has promising activity with a manageable safety profile for patients with inflammatory diseases.


The authors sincerely appreciate the financial supports by Postdoctoral Science Foundation of China (2017M621162), Key Science and Technology Research and Development Plan of Shenyang (17-230-9-05) and College Students Innovation and Entrepreneurship Training Plan of Liaoning (201910164025).


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Figure Caption

Fig. 1 Chemical structure and product spectrum of PH-797804 (A) and the IS (B). Fig. 2 Representative chromatograms of: (A) blank rat plasma, (B) blank rat plasma spiked with PH-797804 (LLOQ) and IS, (C) rat plasma obtained 0.75 h after

Table 1. Precision, accuracy, matrix effect and recovery for analysis of PH-797804 in rat plasma (n = 6)


Matrix effect
RSD (%) RSD (%) (RE%) (%, mean ± SD) (%, mean ± SD)
2.5 8.1 7.0 4.8 95.4 ± 7.5 84.7 ± 6.9
100 6.3 5.7 8.5 94.6 ± 6.7 81.4 ± 6.3
1200 7.7 8.4 -7.8 93.3 ± 6.0 90.2 ± 7.4

Table 2. Stability of PH-797804 in rat plasma (n = 3)

Conditions PH-797804
Spiked (ng/mL) RE (%) RSD (%)
2.5 5.7 7.5
30 days at -20°C 100 6.9 7.0
1200 -4.3 6.2

Three freeze and thaw 2.5

100 3.6

5.3 7.1

2.5 2.9 8.7
12 h at room temperature 100 4.6 6.4
1200 -2.2 4.4

4°C in autosampler for 12 h 2.5

100 3.5

7.6 9.1

in processed samples

Table 3. Pharmacokinetic parameters of PH-797804 with non-compartmental

method (n = 6)

Parameters Units 10 mg/kg 20 mg/kg 40 mg/kg
Cmax* μg/mL 0.65 ± 0.24 1.24 ± 0.26 2.34 ± 0.42
tmax h 2.50 ± 1.22 4.00 ± 1.26 3.67 ± 0.82
t1/2 h 3.69 ± 0.97 3.43 ± 1.08 3.38 ± 1.01

h·μg/mL 4.18 ± 1.46 9.65 ± 1.70 19.27 ± 5.26
h·μg/mL 4.22 ± 1.46 9.83 ± 1.64 19.49 ± 5.32

Vz/F L 14.88 ± 8.02 10.43 ± 4.05 10.66 ± 4.4
CL/F L/h 2.84 ± 1.72 2.08 ± 0.32 2.22 ± 0.74

* 2
AUMC0→24h h ·μg/mL
21.25 ± 7.96
58.65 ± 14.17
121.6 ± 46.3

* 2
AUMC0→∞ h ·μg/mL 22.64 ± 8.07 64.04 ± 16.42 128.2 ± 47.9

⦁ First validated UPLC-MS/MS method for determination of PH-797804 in rat plasma.
The method was applied to a pharmacokinetic study of PH-797804 in rats.
3. The profiles showed a linear relationship between drug concentration and dosage.

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